Phenotypic Characterization of Three Phylogenetically Conserved Stem-Loop Motifs in the Mengovirus 3′ Untranslated Region

Abstract

An alignment of cardiovirus sequences led to the prediction of three conserved stem-loops in the 3′ untranslated region (UTR) of mengovirus. Deletions of each stem were engineered in mengovirus cDNAs and also in mengovirus replicons, in which part of the viral capsid sequences were replaced with the firefly luciferase gene. The effect of deletion on RNA infectivity and plaque phenotype was evaluated after transfection of viral transcripts into HeLa cells or by luciferase assays of cellular extracts after transfection with RNA replicons. Stem I (mengovirus bases 7666 to 7687) was found to be dispensable for viral growth or exponential luciferase expression. Deletion of stem III (bases 7711 to 7721) was lethal to the virus, and the replicons were incapable of RNA synthesis. Deletion of stem II (ΔII; bases 7692 to 7705) produced an intermediate phenotype, in that replicons had marginal RNA synthesis activity but transfection with genomic RNA usually failed to produce plaques after normal incubation times (31 h, 37°C). In a few of the ΔII transfections, however, plaques were observed after long incubation, especially if the cells received large amounts of RNA (3 μg per 3 × 10 ⁶ cells). Viruses from two ΔII-derived plaques were isolated and amplified. Their RNAs were converted into cDNA, sequenced, and mapped for genotype. Each maintained the ΔII deletion and, in addition, had one or two reversion mutations, which were characterized by reverse genetics as responsible for the phenotypes. One reversion caused an amino acid change in the polymerase (3D ^pol ), and the other was localized to the 3′ UTR, upstream of stem I.